长链非编码RNA HULC调控MACC1基因影响脑胶质瘤干细胞特性的机制

There is the relationship between brain glioma stem cells (GSCs) and the genesis, development and recrudescence of brain giloma. We reported firstly that MACC1 gene was up-regulated in human brain glioma tissues. Recent researches found that HULC and MACC1 gene expressed abnormally in GCSs. HULC could regulate the expression of MACC1 gene. The down-regulating of MACC1 gene in GSCs could influence the ability of GSCs to form cell spheres, and increase the sensibility of GSCs to chemotherapy drugs. But the mode and mechanism of regulation are not clear. Combined the researches statuses and our previous work, we presume that HULC up-regulating in GSCs could up-regulate the expression of MACC1 gene as transcription factor; MACC1 protein could interact with FASLG protein, and regulate some stem cell characteristics and biologic behaviors of GSCs by FAS/FASL pathway. There is none related reports about our hypothesis by retrieve. In our study, primer hybridization and luciferase report gene expression analysis are used to identify the direct action and pivotal combined site and regulation mechanism between HULC and MACC1 gene, and ChIP is used to identify the transcription factors which play the regulative action with HULC together; the overexpression and expression silence GSCs of HULC and MACC1 genes are cultured in vitro and in vivo to identify the regulation of HULC to the expression and function of MACC1 gene, and identify the influence of HULC and MACC1 gene to the stem cell characteristics and biologic behaviors of GSCs; we research whether MACC1 can influence the stem cell characteristics and biologic behaviors of GSCs by FAS/FASL pathway; the mode between FASLG and MACC1 and the regulation of FASLG to the function of MACC1 are studied; the inhibitive effects of target down-regulate HULC to glioma eventually. The results of this study can reveal the mechanism of HULC regulating the stem cell characteristics and biologic behaviors of GSCs through MACC1 gene, and provide the theoretical evidence and new target point to the therapy of glioma.

脑胶质瘤干细胞(glioma stem cells，GSCs)与脑胶质瘤发生、恶性生长和复发密切相关。我们首次报道了MACC1基因在人脑胶质瘤组织中表达上调。近期研究发现，HULC和MACC1基因在GSCs中表达异常，HULC能够调控MACC1基因的表达；而且下调GSCs中MACC1基因的表达能够影响GSCs形成细胞球的能力，而且能够提高GSCs的化疗敏感性，但具体作用方式和调控机制不清。本研究通过荧光素酶报告基因等方法，①明确HULC与MACC1基因5'旁侧序列的直接作用、关键结合位点和调节机制；②明确MACC1基因影响脑GSCs干细胞特性的机制；③研究MACC1和FASLG互作的机制，明确FASLG对MACC1功能的调节；④研究靶向下调HULC对胶质瘤的抑瘤效果。本项目的研究结果不仅能揭示脑胶质瘤发生发展的机制，而且能为胶质瘤治疗提供新靶点。

脑胶质瘤是原发性颅内肿瘤，预后差，死亡率高。脑胶质瘤存在肿瘤干细胞(GSCs)，GSCs与脑胶质瘤发生、恶性生长和复发密切相关，对治疗具有更强的抵抗性。对GSCs的深入研究可能为脑胶质瘤的治疗提供新的有效途径。.本项目在前期工作基础上，首先研究HULC与MACC1基因5’旁侧序列的直接作用和关键结合位点，明确HULC对MACC1基因表达的调节作用；其次研究MACC1基因对GSCs的干细胞特性和生物学行为的调控作用；最后研究MACC1是否通过FAS/FASL信号通路调控胶质瘤GSCs的生物学行为和干细胞特性的分子机制，明确HULC和MACC1基因在胶质瘤发生进展中作用。.本研究发现，MACC1是HULC的靶基因，HULC能够在上调MACC1的表达。MACC1基因在脑胶质瘤组织中表达上调，其表达异常与脑胶质瘤的病理分级等临床指标相关，而且能够作为胶质瘤独立的预后因子。MACC1基因能够作为癌基因，促进胶质瘤的恶性表型，沉默其表达能够促进恶性增殖、抑制细胞凋亡，而且可以显著的提高胶质瘤细胞对化疗药物顺铂的敏感性，其发挥上述调节作用主要是通过FAS/FASL信号转导通路实现的。HULC与MACC1基因的表达改变能够显著影响GSCs的成瘤能力，沉默HULC及MACC1基因都能够显著减小GSCs诱导的裸鼠移植瘤的体积，延长荷瘤裸鼠的生存时间。应用免疫共沉淀及Pull-down结合质谱技术，筛选出多个与MACC1蛋白互作的蛋白，如FASL、BMI-1、SIN3A和FUBP1。长链非编码RNA芯片筛查结果分析显示TUSC7、UCA1、HOTTIP及LINC01460等多种lncRNAs存在显著的表达异常。.本项目的研究结果不仅能够揭示HULC通过MACC1基因调控胶质瘤CSCs的生物学行为和干细胞特性的分子机制，为研究脑胶质瘤的发生发展机制提供新的理论依据，而且能够为胶质瘤的治疗提供新靶点。